Gene Cluster of Arthrobacter ilicis Rü61a Involved in the Degradation of Quinaldine to Anthranilate

Abstract

A genetic analysis of the anthranilate pathway of quinaldine degradation was performed. A 23-kb region of DNA from Arthrobacter ilicis Rü 61a was cloned into the cosmid pVK100. Although Escherichia coli clones containing the recombinant cosmid did not transform quinaldine, cosmids harboring the 23-kb region, or a 10.8-kb stretch of this region, conferred to Pseudomonas putida KT2440 the ability to cometabolically convert quinaldine to anthranilate. The 10.8-kb fragment thus contains the genes coding for quinaldine 4-oxidase (Qox), 1H-4-oxoquinaldine 3-monooxygenase, 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, and N-acetylanthranilate amidase. The qoxLMS genes coding for the molybdopterin cytosine dinucleotide-(MCD-), FeSI-, FeSII-, and FAD-containing Qox were inserted into the expression vector pJB653, generating pKP1. Qox is the first MCD-containing enzyme to be synthesized in a catalytically fully competent form by a heterologous host, P. putida KT2440 pKP1; the catalytic properties and the UV-visible and EPR spectra of Qox purified from P. putida KT2440 pKP1 were essentially like those of wild-type Qox. This provides a starting point for the construction of protein variants of Qox by site-directed mutagenesis. Downstream of the qoxLMS genes, a putative gene whose deduced amino acid sequence showed 37% similarity to the cofactor-inserting chaperone XdhC was located. Additional open reading frames identified on the 23-kb segment may encode further enzymes (a glutamyl tRNA synthetase, an esterase, two short-chain dehydrogenases/reductases, an ATPase belonging to the AAA family, a 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase/5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase-like protein, and an enzyme of the mandelate racemase group) and hypothetical proteins involved in transcriptional regulation, and metabolite transport.The genetic diversity and flexibility of prokaryotes has led to the evolution of an impressive variety of metabolic pathways to transform or degrade natural as well as numerous xenobiotic compounds. The genes coding for enzymes involved in degradative pathways are often organized as operons and supraoperonic clusters comprising "pathway modules" (1-4).N-Heteroaromatic compounds are metabolized and even mineralized by various bacteria (for a review, see Ref. 5 and references therein). Quinaldine (2-methylquinoline) is utilized by Arthrobacter ilicis Rü 61a as a source of carbon, nitrogen, and energy; its degradation proceeds via the "anthranilate pathway" (5, 6). The initial step, the hydroxylation of quinaldine in para position to the N-heteroatom, is catalyzed by the inducible enzyme quinaldine 4-oxidase (Qox). 1 Qox is a molybdo-iron/sulfur-flavoprotein with an (LMS) 2 subunit structure and has been classified to belong to the xanthine oxidase family of molybdenum enzymes (7-10; for reviews on molybdenum enzymes, see Refs. 11-13). Like many other bacterial molybdenum hydroxylases, e.g. quinoline 2-oxidoreductase (Qor) from Pseudomonas putida 86 (14, 15), isoquinoline 1-oxidoreductase (Ior...

Regioselective aromatic hydroxylation of quinaldine by water using quinaldine 4-oxidase in recombinant Pseudomonas putida

“…Recombinant qoxLMS gene expression in Pseudomonas strains was achieved by means of the plasmid pKP1 [38], a qoxLMS-containing derivative of the broad-host-range cloning vector pJB653. The expression of qoxLMS is regulated by the plasmid-encoded XylS protein which activates the P m promoter-mediated expression in the presence of XylS effectors such as benzoate or 2-methylbenzoate.…”

Transcriptional Regulation of the ant Operon, Encoding Two-Component Anthranilate 1,2-Dioxygenase, on the Carbazole-Degradative Plasmid pCAR1 of Pseudomonas resinovorans Strain CA10

“…meta-Cleavage enzyme CarB is a heterotetrameric enzyme (␣ 2 ␤ 2 ) composed of CarBa and CarBb subunits. The resulting metabolite, anthranilate, is a naturally occurring compound formed through tryptophan degradation (19) and is also known as an important intermediate in the metabolism of many N-heterocyclic compounds, such as indole (15,21,23), o-nitrobenzoate (5,10), and quinaldine (28). Via dioxygenation catalyzed by class IB (2) two-component anthranilate 1,2-dioxygenase (AntDO) composed of the terminal oxygenase component (AntAB) and the reductase component (AntC), followed by spontaneous deamination and decarboxylation, anthranilate is converted to catechol, which is further degraded to tricarboxylic acid (TCA) cycle intermediates (25,26).…”

Integrated organic–aqueous biocatalysis and product recovery for quinaldine hydroxylation catalyzed by living recombinant Pseudomonas putida

“…Bacterial strains, plasmid, and chemicals Pseudomonas putida KT2440 [2] and plasmid pKP1 [26] were used as the recombinant host strain and the expression vector, respectively, for the functional expression of qoxLMS genes. Media components and other chemicals were obtained from Merck (quinaldine, C98%; Hohenbrunn, Germany), Sigma-Aldrich (4-hydroxyquinaldine, C98%; Steinheim, Germany), or Fluka (1-dodecanol, 98.5%; Buchs, Switzerland).…”